Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods

Abstract

Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman’s rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.

Detection of Klebsiella pneumoniae in healthy poultry: Insights and perspectives from culturing and metagenomics

“…In contrast to a study on K. pneumoniae in human faecal samples (Lindstedt et al, 2022), the relative abundance of K. pneumoniae in the metagenomes did not correlate well with the culture quantification. The combination of the potentially un-culturable K. pneumoniae and the DNA extraction kit not being optimal for poultry cecal samples may have had an impact on these results.…”

“…To date, WMS is the most comprehensive method for the classification of the gut microbiota. WMS, qPCR and culturing of K. pneumoniae have previously been compared with success on human faecal samples (Lindstedt et al, 2022), but no studies have so far compared these methods for the detection and quantification of K. pneumoniae in samples from other hosts, such as broilers and turkeys.…”

Modelling the Gastrointestinal Carriage of Klebsiella pneumoniae Infections

“…S2 ). Kp52145 was detected by a qPCR method that enabled the specific detection of K. pneumoniae by amplifying the intergenic region between the zur and khe genes, named ZKIR for the zur-khe intergenic region ( 23 , 24 ). No significant differences were observed in the alpha diversity, independent of the test used to measure it ( Fig.…”

“…The DNA samples used for the microbiome analysis were analyzed via the ZIKIR-qPCR method, which has previously been described in the detection of the presence of K. pneumoniae ( 23 , 24 ). DNA was amplified in a 5 μL PCR mix that contained 0.4 μL of each ZKIR primer (final concentration, 200 nM each) ( Table S2 ), 2.6 μL of KAPA SYBR FAST qPCR mix, and 2 μL of sample DNA.…”

Comparative Analysis of Complicated Urinary Tract Infections Caused by Extensively Drug-Resistant Pseudomonas aeruginosa and Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae

“…Our results could be partly explained by the worse eradication and recurrence rates observed in the ESBL -K. pneumoniae group and thus, higher hospital readmission rates. While K. pneumoniae is a normal part of the intestinal flora in humans, hospital patients are more susceptible to colonization by this bacterium, which further complicates eradication [ 58 ]. Another possible explanation is that the patients with the highest prevalence of ESBL- K.pneumoniae cUTI were kidney transplant recipients and those with chronic renal failure.…”